Syntheses and Investigations of Conformationally Restricted, Linker-Free α-Amino Acid–BODIPYs via Boron Functionalization

A series of α-amino acid–BODIPY derivatives were synthesized using commercially available N-Boc-l-amino acids, via boron functionalization under mild conditions. The mono-linear, mono-spiro, and di-amino acid–BODIPY derivatives were obtained using an excess of basic (histidine, lysine, and arginine), acidic (aspartic acid), polar (tyrosine, serine), and nonpolar (methionine) amino acid residues, in yields that ranged from 37 to 66%. The conformationally restricted mono-spiro- and di-amino acid–BODIPYs display strong absorptions in the visible spectral region with high molar extinction coefficients and significantly enhanced fluorescence quantum yields compared with the parent BF2–BODIPY. Cellular uptake and cytotoxicity studies using the human HEp2 cell line show that both the presence of an N,O-bidentate spiro-ring and basic amino acids (His and Arg) increase cytotoxicity and enhance cellular uptake. Among the series of BODIPYs tested, the spiro-Arg- and spiro-His-BODIPYs were found to be the most cytotoxic (IC50 ∼ 22 μM), while the spiro-His-BODIPY was the most efficiently internalized, localizing preferentially in the cell lysosomes, ER, and mitochondria.


■ INTRODUCTION
Boron dipyrrin or boron dipyrromethene (BODIPY) dyes 1−6 are a class of boron-coordinated organic dyes that display a multitude of highly desirable properties for various applications, including good solubility in various solvents, large molar extinction coefficients, high fluorescence quantum yields, relatively sharp absorption and emission bands, low cytotoxicity, and structural tunability. With these extraordinary properties, BODIPY dyes have been widely applied in, for example, live-cell bioimaging, 7,8 photodynamic therapy, 9,10 fluorescent sensing, 11 dye-sensitized solar cells, 12 and viscosity detection. 13 Several methodologies for the post-functionalization of BODIPYs have been developed over the past 2 decades. 12,14 The absorption and emission properties of BODIPY dyes can be fine-tuned from ca. 400 15,16 to 750 nm through functionalization reactions at the dipyrrin core. 14 In contrast, modifications at the boron center generally have little or no effect on the absorption and emission wavelengths, although the 3D structures of the boron-functionalized BODIPY molecules often change significantly upon the introduction of various groups at the boron atom. As a result, the fluorescence quantum yields, laser efficiencies, 17 redox behavior, aqueous solubility, aggregation behavior, and chemical stability 18−20 of BODIPYs can be conveniently fine-tuned for specific applications. Nucleophilic substitution of one or both fluorides at the boron position provides the possibility for application in positron emission tomography−optical dual imaging by replacing the nonradiative fluorine atom(s) by 18 F. 21 Other investigations of C-, O-, and N-BODIPYs 22−25 have led to enhanced materials for applications in energy-transfer cassettes, fluorescence imaging, and chiral recognition, among others. However, only a few reports have explored bidentate B-spiro-BODIPYs. 26−28 Amino acid−fluorophore conjugates are important building blocks for the construction of bioactive fluorescent peptides and proteins. The synthesis of BODIPY−amino acid conjugates has been reported through C−H activation 29,30 and nucleophilic aromatic substitution reactions. 31 We have previously reported the synthesis of N,O-bidentate spiro-Gly-BODIPY derivatives by direct functionalization at the boron atom 32 and extended this methodology to the preparation of near-IR aza-BODIPY-Gln derivatives with potential application in photodynamic therapy. 33 In the past few decades, the biological activities of boroncontaining compounds have been investigated for their antifungal, antibacterial, antiviral, anti-inflammatory, and antiprotozoal activities. 34−37 Notably, tavaborole (Kerydin), favor the formation of conformationally restricted products B and C. The tert-butyloxycarbonyl (Boc) electron-withdrawing group on the N-terminus of the amino acid is necessary for decreasing the charge on boron, therefore leading to stable N(sp 3 ) conjugates. Some amino acids bearing nucleophilic side chains also required the use of side chain protecting group(s) to avoid byproduct formation via a side chain attack on the boron atom. However, the widely used 9-fluorenylmethyloxycarbonyl (Fmoc) and trifluoroacetyl protecting groups require harsh conditions for deprotection, 42 which could also remove the N-Boc group and/or cause BODIPY degradation. 20 Therefore, the carboxylbenzyl (Cbz) or benzyl (Bn) groups were employed to protect the amino acids bearing nucleophilic side chains. The advantages of these two protecting groups are as follows: (1) the Cbz and Bn deprotection can be achieved under mild, neutral conditions in quantitative yields; (2) this strategy eliminates the formation of byproducts during the linker-free conjugation reaction; (3) the generated toluene product from Cbz or Bn deprotection is easily removed via vacuum evaporation; (4) Cbz or Bn protection can be applied to a variety of O,N-nucleophilic side chains; and (5) deprotection of the Cbz or Bn group does not affect the Boc group on the nitrogen adjacent to boron. Catalytic hydrogenation was performed to deprotect the Cbz or Bn side group(s) of the BODIPY derivatives (Scheme 1). The disappearance of the benzyl group (2H at ca. 5.0 ppm and 5H at ca. 7.5 ppm in CDCl 3 ) could be clearly seen by 1 H NMR, as shown in the Supporting Information. This methodology was successfully applied to the Asp-, Arg-, His-, and Ser-BODIPY conjugates, producing the corresponding a− c analogues. However, in the case of the Lys-BODIPY derivatives 9A−C, upon the removal of the Cbz protecting group, the products decomposed within a few hours at room temperature, likely due to a nucleophilic attack of the amine upon the boron atom. The Arg-BODIPY was also observed to slowly decompose within 24 h; however, the His-BODIPY and all other derivatives bearing acidic, polar, and nonpolar amino acid side chains are stable in both solution and solid forms for at least 1 week at room temperature.
Presumably, the reaction of BODIPY 1 in the presence of boron trichloride proceeds via either the formation of a boronium cation intermediate, 41 followed by the nucleophilic attack of the amino acid carboxylate group on boron to produce product A, or via the in situ formation of a Cl 2 -BODIPY intermediate 43−45 leading to products B and C.
The above methodology was extended to the synthesis of BODIPY derivative 8 bearing an azide-functionalized lysine residue suitable for further conjugation with alkyne-containing compounds via click chemistry. In addition, the tripeptide, N-Boc-GlyGlyGly was also prepared using this methodology, as shown in Scheme 2, producing N,O-bidentate (Gly) 3 -BODIPY 11B as the major product in 26.0% yield, followed by 11C in 19.5% yield.
The structures of all α-amino acid-BODIPY conjugates were confirmed by 1 H, 13 C, 11 B-NMR, and high-resolution mass spectroscopy (HRMS) (see the Supporting Information). In all cases, the 9H in the Boc protecting groups can be seen at 1.4 ppm in the 1  Computational Studies. Our previous computational and NMR studies 32 showed that the spiro-Gly-BODIPY derivatives exist as two conformers (up and down) at room temperature, depending on the orientation of the Boc group with respect to the meso-phenyl, with the up-conformer being slightly more stable (ΔG < 3 kcal/mol). A similar trend is observed in the current series of amino acid-BODIPY derivatives. All the calculated data shown in Table S1 of the Supporting Information are for the up-conformers of the respective N,Obidentate BODIPYs B. The mono-(A) and di-(C) amino acid−BODIPYs also exist in the form of two conformers with small energy differences (ΔG < 5 kcal/mol). All data discussed below refer to the more stable conformers.
Analysis of the calculated relative Gibbs energies of BODIPYs 2a−7a, 2b−7b, and 2c−7c in dichloromethane demonstrates that the mono-substituted a is the most stable of the three forms, while the conformationally restricted spirocompound b and the di-substituted c have similar Gibbs energies (see Table S1). The effect of the side chain is small and the energy differences range between 14 and 19 kcal/mol. However, experimentally BODIPYs bearing amino acids with strongly basic side chains, such as Arg and Lys, slowly decomposed in solution over a 24 h period, while all others, including His-BODIPY, were stable in solutions for over 1 week at room temperature. These results are consistent with previous observations of BODIPY instability under strongly basic conditions, such as in the presence of t BuOK 46 or NH 4 OH. 18 The lower stability of the di-substituted BODIPYs c compared with the mono-derivatives a is mostly due to entropy effects. On the other hand, the lower stability of the N,O-bidentate BODIPYs b is probably due to their relatively weaker B−N(Boc) bond compared with the B−O and B−F bonds. The B−N(Boc) bonds range between 1.541 and 1.547 Å with 3b having the longest bond of the series (see Table S1). The B−N and B−O bond lengths are similar in all the investigated N,O-bidentate spiro-BODIPYs and do not change significantly when different amino acids are introduced. However, for all compounds in the series, the B−N(dipyrrin) bonds in the spiro-BODIPYs b are consistently 0.01−0.02 Å longer than the ones in the mono a and di-substituted c BODIPYs. Our previous studies on the stability of BODIPYs 16 suggest that the spiro-conjugates will therefore be slightly less stable than the di-substituted compounds. Furthermore, our calculations show that the N,O-bidentate b compounds are consistently more polar than the mono a and di-substituted c analogues throughout the entire series, with 2b spiro-Asp being the most polar of all (see Table S1). The higher polarity of the N,O-bidentate spiro-compounds explains their observed higher solubility in polar solvents.
Spectroscopic Properties. The spectroscopic properties of the selected conformationally restricted BODIPYs 2b,c, 3b,c, 4b,c, 5B,C, 6B,C, and 7b,c were measured and calculated in acetonitrile. The experimental results are shown in Table 1, and the calculated parameters are shown in the Supporting Information, Table S2. The bidentate spiro-(B) and the di-(C) amino acid-BODIPY derivatives have similar absorption and fluorescence profiles, with maximum absorption and emission bands centered at ca. 503 and 513 nm, respectively. These maximum absorption and emission wavelengths are slightly red-shifted (4−5 nm) compared with the parent BF 2 -BODIPY 1. This shift is in agreement with the performed TD-DFT calculations (Table S2). The predicted red shift is 3−5 nm and is due to the slightly smaller HOMO−LUMO gap for the αamino acid−BODIPYs.
The similar absorption and emission profiles observed for this series of α-amino acid−BODIPYs are consistent with the calculated similar HOMO−LUMO energies and gaps, as shown in Figure 1 and the Supporting Information, Figure   S103. Figure 1 represents the frontier orbitals for the N,Obidentate spiro and di-substituted BODIPYs 6B and 6C. Both HOMO and LUMO are localized on the BODIPY core with no significant contribution from the amino acid nor the Boc protecting group. This is also consistent with our previous findings 32 and is observed for all other α-amino acid− BODIPYs in the series. The performed TD-DFT calculations show that HOMO → LUMO is the leading transition for all BODIPYs studied. Given the fact that the forms and energies of these two orbitals are essentially independent of the amino acid, it is not surprising that the maximum absorption and emission wavelengths are very similar among the entire series.
Remarkably, all newly synthesized α-amino acid−BODIPYs display higher fluorescence quantum yields (Φ ∼ 0.9) compared with the parent BF 2 −BODIPY 1 (Φ = 0.61), making them promising candidates for fluorescence labeling of bioactive peptides. As also suggested by our previous findings, 32 this result is probably due to the increased rigidity around the boron center in these conformationally restricted BODIPYs, which minimizes vibrations in-and-out of the dipyrrin plane. Interestingly, the calculated oscillator strengths of all α-amino acid−BODIPYs are smaller than that of parent BF 2 -BODIPY 1. However, the increased rigidity around the boron center most likely reduces the nonradiative vibrational relaxation, resulting in increased quantum yields.
Cellular Properties. Cytotoxicity. The cytotoxicity of the selected conformationally restricted α-amino acid−BODIPY 2b,c, 3b,c, 4b,c, 5B, 6B, and 7b were evaluated in human carcinoma HEp2 cells exposed to the increasing concentrations of each BODIPY up to 200 μM; the results are shown in the Supporting Information, Figures S104 and S105 and summarized in Table 2. Among the series ( Figure S105), the spiro-Arg 3b and spiro-His 4b are the most toxic BODIPYs with IC 50 values of 22 and 23 μM, respectively. These results indicate that both a spiro-5-membered ring on boron (as often observed in boron-based therapeutics, such as spiro-borates), and the presence of basic amino acids, such as Arg and His, increase the cytotoxicity of the compound. Indeed, a 3−5-fold increase in cytotoxicity was observed for the spiro-BODIPYs in comparison with the corresponding di-amino acid derivatives ( Figure S105), possibly due to the more rigid N,O-bidentate  Cellular Uptake and Intracellular Localization. The time-dependent cellular uptake of the most cytotoxic basic αamino acid BODIPYs 3b and 4b and of the parent BODIPY 1 at a nontoxic concentration of 10 μM were evaluated in human carcinoma HEp2 cells (Figure 2). The basic amino acids Arg and His are able to interact with phosphate groups on plasma membranes, which enhances their cellular uptake. BODIPY 4b containing His showed the highest uptake, about 2-fold that of the parent BODIPY 1 at times >2 h. The lower uptake observed for the Arg-BODIPY 3b, compared with 4b at all the times investigated, might in part be a result of its relatively lower stability due to the greater basicity of the guanidinium group. Although at times <8 h, BODIPY 3b showed a lower uptake than parent BODIPY 1, the positively charged guanidinium group in 3b favored its continuous uptake over time via interaction with the negatively charged plasma membrane, thus leading to an enhanced uptake at 24 h relative to BODIPY 1. 48,49 The sub-cellular localization of BODIPYs 3b ( Figure 3) and 4b ( Figure 4) were also investigated by fluorescence microscopy upon the exposure of the HEp2 cells to 10 μM of each BODIPY for 6 h. Overlay experiments using the organelle-specific fluorescent probes LysoTracker deep red (lysosomes), MitoTracker deep red (mitochondria), ER Tracker blue/white (ER), and Hoechst 33342 (nuclei) were conducted to evaluate the preferential sites of BODIPY localization. The results obtained are shown in Figures 3 and  4. Both BODIPYs 3b and 4b are found to localize in the ER, mitochondria, and lysosomes. We have previously reported that BODIPY 1 accumulates preferentially in the ER, Golgi, and lysosomes. 19 Our results suggest that BODIPYs bearing positively charged amino acids (Arg, His) also localize in mitochondria, which might account for their observed higher cytotoxicity.

■ CONCLUSIONS
A series of conformationally restricted α-amino acid− BODIPYs, bearing basic (His, Lys, and Arg), acidic (Asp),   The conformationally restricted amino acid−BODIPYs display similar absorption and emission properties, slightly red-shifted relative to the parent BF 2 −BODIPY due to their slightly smaller HOMO−LUMO gap, and TD-DFT calcu-lations suggest that HOMO → LUMO is the leading transition. Furthermore, all conformationally restricted amino acid−BODIPYs showed enhanced fluorescence quantum yields (Φ ∼ 0.9) compared with the parent BF 2 −BODIPY (Φ = 0.61) due to the increased rigidity around the boron center, which minimizes vibrations in-and-out of the dipyrrin plane.
Investigations in human HEp2 cells showed that the N,Obidentate spiro-Arg and spiro-His are the most cytotoxic (IC 50 ∼ 22 μM), 3−5-fold more toxic than their corresponding diamino acid BODIPY derivatives. The basic amino acid− BODIPYs are able to interact with negatively charged groups on plasma cell membranes showing an enhanced cellular uptake relative to the parent BF 2 −BODIPY at 24 h. The preferential sites of intracellular localization for the most cytotoxic basic amino acid−BODIPYs were found to be the ER, mitochondria, and lysosomes, which might account for their observed higher cytotoxicity.

■ EXPERIMENTAL SECTION
General. Commercial grade chemical reagents were purchased from VWR or Sigma-Aldrich and used without further purification. Liquid column chromatography with silica gel (60 Å, 230−400 mesh) or preparative TLC plates (60 Å, 20 × 20 cm 2 , 210−270 μm) were used for all the purifications. NMR spectra were collected using a Bruker AV-400 or AV-500 spectrometer at 300 K (operating at 400 or 500 MHz for 1  where Φ X and Φ ST are the quantum yields of the sample and standard, Grad X and Grad ST are the gradients from the plot of the integrated fluorescence intensity versus absorbance, and η represents the refractive index of the solvent. BODIPY 1 was prepared as previously reported in the literature. 41 Larger scale syntheses of A−C products have been previously reported. 33 Computational Methods. The geometries of all compounds and complexes were optimized without symmetry constraints using the B3LYP/6-31+G(d,p) level. 50−52 The solvent effects were taken into account using the polarized continuum model. 53,54 The stationary points on the potential energy surface were confirmed with frequency calculations. The UV−vis absorption data were calculated using the TD-DFT method 55 All calculations were performed using the Gaussian 09 program package. 56 Cell Culture. Cytotoxicity. All the cell culture media and reagents were purchased from VWR and fisher scientific. The human carcinoma HEp2 cells were purchased from ATCC and maintained in a MEM containing 5% FBS and 1% penicillin/streptomycin antibiotic. The cells were then sub-cultured and maintained twice weekly. Cell toxicity was evaluated using the CellTiter-Blue (Promega) assay.
The HEp2 cells were plated at 7500−10 000 cells per well in a Costar 96-well plate and allowed to grow for 24−48 h. BODIPY samples were dissolved in 100% DMSO to prepare 32 mM stock The Journal of Organic Chemistry pubs.acs.org/joc Article solutions. Then, a 400 μM working solution was prepared for each sample. Through a 2-fold dilution procedure, HEp2 cells were exposed to concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 μM solutions and incubated for 24 h at 37°C. The working solutions were then removed, and the cells were washed with 1 × PBS buffer triple times. The cells were exposed to the medium containing 20% CellTiterBlue and incubated for 4 h at 37°C. The viability of the cells is measured by reading the fluorescence of the medium at 570/615 nm using a BMG FLUOstarOptima microplate reader. The fluorescence signal of the untreated cells in media only was normalized to 100%. Cellular Uptake. Costar 96-well plates were prepared as described above. Each BODIPY solution (10 μM) was added to the cells and incubated for 0, 1, 2, 4, 8, and 24 h. At the end of the incubation period, the BODIPY solution was removed, and the cells were washed with 1 × PBS buffer triple times, followed by solubilizing the cells with 0.25% Triton X-100 in 1 × PBS buffer. The compound standard curve of each BODIPY was prepared by using BODIPY solutions with concentrations of 0, 0.31, 0.63, 1.25, 2.5, 5, and 10 μM in 1 × PBS buffer containing 0.25% X-100. The standard curve of the cell number was prepared using 10 000, 20 000, 40 000, 60 000, 80 000, and 100 000 cells per well and quantified by using a CyQuant Cell Proliferation Assay. The BODIPY concentration in cells at the end of each incubation period was determined using a BMG FLUOstar Optima microplate reader at the excitation wavelength and emission wavelength of each BODIPY. The time-dependent cellular uptake was evaluated as nM/cell.
Microscopy. The human HEp2 cells were incubated in a 35 mm tissue culture dish (CELLTREAT) and allowed to grow overnight. The cells were exposed to each compound at a concentration of 10 μM and incubated for 6 h (5% CO 2 , 37°C). The following organelle trackers (Invitrogen) were added to the cells: ER Tracker Blue/White (1 mM), Hoechst 33342 (16 mM), MitoTracker deep red (1 μM), and LysoSensor deep red (1 μM). The cells were incubated with each compound and trackers for half an hour, washed with PBS solution three times, and followed by fixing with 4% paraformaldehyde before imaging. A Leica DM6B upright microscope equipped with a 40 × water immersion objective, and DAPI, GFP, and Texas Red filter cubes (Chroma Technologies) were used to acquire the images.
Synthesis. General Procedure for the Preparation of α-Amino Acid−BODIPY Derivatives. BODIPY 1 (50 mg, 0.154 mmol) was dissolved in 5 mL of dry dichloromethane, followed by the addition of 2 equivalents of BCl 3 (1 M in toluene, 0.3 mL, 0.308 mmol). The reaction was stirred for 1 h at room temperature. Subsequently, a few drops of dry triethylamine were added to the reaction mixture. The Nprotected amino acids (4 equivalents) were dissolved in a vial with dry dichloromethane and a few drops of dry triethylamine. The amino acid solutions were then added dropwise and stirred at room temperature for 1 h. The reaction mixture was then poured into a saturated NaHCO 3 solution and extracted with dichloromethane (10 mL × 3). The organic layers were combined and dried over anhydrous Na 2 SO 4 . The organic solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel and recrystallized to afford the desired BODIPYs.